Functional analysis of 150 deletion mutants in Saccharomyces cerevisiae by a systematic approach.

In a systematic approach to the study of Saccharomyces cerevisiae genes of unknown function, 150 deletion mutants were constructed (1 double, 149 single mutants) and phenotypically analysed. Twenty percent of all genes examined were essential. The viable deletion mutants were subjected to 20 different test systems, ranging from high throughput to highly specific test systems.

Yeast cells as tools for target-oriented screening.

Information about biomolecular interaction networks is crucial for understanding cellular functions and the development of disease processes. Many diseases are known to be based on aberrations of DNA sequences encoding proteins with key functions in the cellular metabolism. Alterations in the respective proteins often lead to disturbances in biomolecular interactions caused by unbalanced stoichiometries, and thus result in alterations of molecule fluxes, cell architecture and signalling pathways.

Single-read sequence tags of a limited number of genomic DNA fragments provide an inexpensive tool for comparative genome analysis.

Single-read sequences from both ends of 415 3-kb average size genomic DNA fragments of Candida albicans were compared with the complete sequence data of Saccharomyces cerevisiae. Comparison at the protein level, translated DNA against protein sequences, revealed 138 sequence tags with clear similarity to S. cerevisiae proteins or open reading frames.

Drug-induced phenotypes provide a tool for the functional analysis of yeast genes.

The post-genome sequencing era of Saccharomyces cerevisiae is defined by the analysis of newly discovered open reading frames of unknown function. In this report, we describe a genetic method for the rapid identification and characterisation of genes involved in a given phenotype. This approach is based on the ability of overexpressed genomic DNA fragments to cure an induced phenotype in yeast.

Homologous recombination partly restores the secretion defect of underglycosylated acid phosphatase in yeast.

The majority of secreted acid phosphatase in Saccharomyces cerevisiae is encoded by the PH05 gene. The secretion level of this acid phosphatase is directly determined by its level of glycosylation. Consequently, PHO5-11-encoded acid phosphatase which lacks 11 of 12 glycosylation sites is only poorly secreted.

Biochemical properties and excretion behavior of repressible acid phosphatases with altered subunit composition.

Yeast repressible acid phosphatase (rAP) is the oligomeric extracellular enzyme encoded by the three structural genes PH05 (p60), PHO10 (p58) and PHO11 (p56). We examined the ability of acid phosphatases formed by various subunit combinations to be excreted into the medium. Plasmids with repressible acid phosphatase structural genes under control of the yeast glyceraldehyde-phosphate dehydrogenase (GAP) promoter were constructed to obtain constitutive expression of acid phosphatase, and yeast strains with disruptions in PHO5, PHO10 and PHO11, respectively, were used to generate mutants expressing single genes or specific gene combinations.

High-level expression of endogenous acid phosphatase inhibits growth and vectorial secretion in Saccharomyces cerevisiae.

The secretion pathway of Saccharomyces cerevisiae was challenged by constitutively overexpressing plasmid-encoded acid phosphatase, a secreted endogenous glycoprotein. A 2-microns-based multicopy plasmid carrying the coding sequence of acid phosphatase under the control of a truncated variant of the strong constitutive glyceraldehyde-3-phosphate dehydrogenase promoter was used for expression. Selection for the promoterless dLEU2 marker leads to a growth arrest.

Identification of the yeast ACC1 gene product (acetyl-CoA carboxylase) as the target of the polyketide fungicide soraphen A.

Soraphen A, a polyketide isolated from the myxobacterium Sorangium cellulosum, is a potent inhibitor of fungal growth. We have used a genetic approach to localize the target of this drug, employing Saccharomyces cerevisiae as a model organism. we have isolated soraphen A-resistant mutants and found that all of them map at the same genetic locus and exhibit a broad range of semidominant phenotypes.

Characterization of the Aspergillus niger pelB gene: structure and regulation of expression.

The nucleotide sequence of pelB, a member of the Aspergillus niger pectin lyase multigene family, has been determined. The pelB gene product, PLB, shares 65% amino acid identity with pectin lyase A (PLA) and 60% with pectin lyase D (PLD). Although growth of pelB multicopy transformants on pectin-containing media results in elevated pelB mRNA levels, pectin lyase B (PLB) is barely detectable.

Temperature sensitivity of the cdc9-1 allele of Saccharomyces cerevisiae DNA ligase is dependent on specific combinations of amino acids in the primary structure of the expressed protein.

In this study we present the characterization of the temperature-sensitive mutant allele cdc9-1 encoding DNA ligase, of Saccharomyces cerevisiae strain A364A by DNA sequencing. Comparison with the published wild-type sequence from strain SK1 revealed 13 nucleotide exchanges between these two sequences, which are derived from non-isogenic genetic backgrounds. Only four of these changes, distributed over the whole coding region, lead to amino acid exchanges in the protein chain.

Heterologous expression of human microsomal epoxide hydrolase in Saccharomyces cerevisiae. Study of the valpromide-carbamazepine epoxide interaction.

A cDNA of human microsomal epoxide hydrolase (hmEH) was constitutively and inducibly expressed in Saccharomyces cerevisiae. The heterologous enzyme was located mainly in the microsomal fraction of yeast cells. Yeast microsomes containing hmEH exerted styrene oxide hydrolase activity (Km = 300 microM; Vmax = 22 nmol/mg min) as well as carbamazepine epoxide hydrolase activity.

Removal of N-glycosylation sites of the yeast acid phosphatase severely affects protein folding.

The influence of N glycosylation on the production of yeast acid phosphatase was studied. A set of synthetic hypoglycosylation mutants was generated by oligonucleotide-directed mutagenesis of the 12 putative sequons (Asn-X-Ser/Thr). Derepression of the hypoglycosylation mutants and analysis of their molecular sizes showed that all 12 sequons of the wild-type acid phosphatase are glycosylated.

A new system for amplifying 2 microns plasmid copy number in Saccharomyces cerevisiae.

The yeast 2 microns plasmid is found in the nucleus of almost all Saccharomyces cerevisiae strains. Its replication is very similar to that of chromosomal DNA. Although the plasmid does not encode essential genes it is stably maintained in the yeast population and exhibits only a small, though detectable, loss rate.

The yeast phosphatase system.

Yeast cells produce a set of enzymes which are involved in the metabolism of phosphate, and include acid and alkaline phosphatases as well as permeases. Most of these enzymes are synthesized in response to the presence or absence of inorganic phosphate. In the past few years a considerable amount of genetic and molecular evidence has accumulated and a rather precise overall picture emerges which describes the mechanism of phosphate control at the level of gene activation.

Constitutive and inducible expression of human cytochrome P450IA1 in yeast Saccharomyces cerevisiae: an alternative enzyme source for in vitro studies.

A cDNA of human cytochrome P450IA1 was expressed in yeast Saccharomyces cerevisiae on a multicopy plasmid under the control of the constitutive GAPFL or the inducible PHO5 promoter. Microsomes of transformed yeast contained substantial amounts of the heterologous enzyme as determined by reduced CO-difference spectra (156-68 pmol/mg). Enzyme kinetics with 7-ethoxyresorufin as substrate resulted in a Km value of 92 nM and a Vmax value of 223 pmol/mg/min, which is comparable to data obtained with human liver microsomes.

The influence of GAP promoter variants on hirudin production, average plasmid copy number and cell growth in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has been engineered to synthesize and secrete desulfato-hirudin (hirudin), a thrombin inhibitor from the leech Hirudo medicinalis. The synthetic gene coding for hirudin was expressed constitutively under the control of four size-variants of the yeast glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) and cloned into a 2 mu based multicopy yeast vector. The constitutive action of the four promoter variants was confirmed by demonstrating that the expression and secretion of hirudin is growth-related.

In vivo translocation of the cell wall acid phosphatase across the yeast endoplasmic reticulum membrane: are there multiple signals for the targeting process?

The repressible Saccharomyces cerevisiae acid phosphatase (APase) coded by the PHO5 gene is a cell wall protein that follows the yeast secretory pathway. We had previously described the in vivo fate of a multicopy plasmid-encoded modified protein, lacking 15 out of 17 signal peptide amino acids. This modified protein accumulates mainly within the cell as an inactive unglycosylated form.

Functional analysis of the signal-sequence processing site of yeast acid phosphatase.

A systematic study of the signal peptidase cleavage site of the main cell-wall-repressible Saccharomyces cerevisiae acid phosphatase encoded by the PHO5 gene is presented. The last amino acid of the signal sequence, the chromosomally encoded alanine of the wild-type gene, was changed by any of 19 other amino acids in the chromosomal DNA by using in vitro mutagenesis in Escherichia coli and the technique of gene replacement. Processing and secretion are normal when the amino acid at this position is a small neutral amino acid, i.

The two positively acting regulatory proteins PHO2 and PHO4 physically interact with PHO5 upstream activation regions.

The repressible acid phosphatase gene PHO5 of Saccharomyces cerevisiae requires the two positively acting regulatory proteins PHO2 and PHO4 for expression. pho2 or pho4 mutants are not able to derepress the PHO5 gene under low-Pi conditions. Here we show that both PHO2 and PHO4 bind specifically to the PHO5 promoter in vitro.

Interpathway regulation of the TRP4 gene of yeast.

Two regulatory proteins, PHO2 and the general control regulator GCN4, bind in vitro to the promoter of the tryptophan biosynthetic TRP4 gene; the TRP4 gene product catalyses the phosphoribosylation of anthranilate. PHO2 binds specifically to the TRP4 promoter, but does not bind to any other TRP promoter. PHO2 and GCN4 proteins bind in a mutually exclusive manner to the same sequence, UAS1, one of two GCN4 binding sites in the TRP4 promoter.

A 28-bp segment of the Saccharomyces cerevisiae PHO5 upstream activator sequence confers phosphate control to the CYC1-lacZ gene fusion.

Two regions within the Saccharomyces cerevisiae PHO5 upstream activator sequence (UAS) are involved in phosphate dependent transcription activation [Rudolph and Hinnen, Proc. Natl. Acad.